Approximately 44,000 women die of breast cancer in the United States each year, and after lung cancer, breast cancer is the most common cause of cancer death in women. Several aspects of breast cancer onset and disease progression have been linked to members of the TGF2 superfamily and their associated downstream signaling components, the Smads. The long-term goal of our work is to investigate how alterations in Smad 3 signal transduction affect breast cancer progression to help establish a molecular staging of disease and to facilitate the discovery of novel treatment strategies that will result in disease regression. The hypothesis of this proposal is that in cyclin overexpressing breast cancers, activation of CDK4/2 leads to phosphorylation and inhibition of Smad 3, thus releasing cell cycle arrest and promoting cell proliferation and metastasis. Our hypothesis is based on the following observations: 1) Smad 3 signaling contributes to G1 cell cycle arrest through transcription of cyclin dependent kinase (CDK) inhibitors and repression of the cell cycle mitogen c-myc, 2) Smad 3 action is inhibited upon phosphorylation by CDK4/2, kinases that are regulated by cyclins D and E, 3) Certain aggressive, basal-type breast cancers overexpress cyclins and have poor outcomes associated with highly metastatic disease. Predicated on these findings, the three specific aims of this proposal will directly examine the interaction between cyclin overexpression and Smad 3 inhibition in breast cancer as follows: Aim 1: Investigate cyclin-mediated mechanisms of Smad 3 inhibition and the impact of this inhibition on G1 arrest. This aim will test the effects of CDK phosphorylation on Smad 3-mediated cell cycle control and utilize a transcription factor reporter array to assess the downstream consequences of cyclin overexpression in breast cancer cells. Aim 2: Investigate the effect of CDK inhibition on the proliferation of cyclin overexpressing breast cancer cells in vitro and in primary and metastatic xenografts using murine models. This aim will test the hypothesis that CDK4/2 inhibition of Smad 3 phosphorylation decreases breast cancer cell proliferation in in vitro cultures and in vivo murine models. Aim 3: Investigate the expression patterns of Smad 3, cyclin D, cyclin E, CDK4 and CDK2 in grades 1, 2, and 3 human breast cancer tissues, basal-like breast cancers, and normal mammary tissue. This aim will test whether subtypes of breast cancer exist with patterned expression of Smad 3, cyclins and CDKs, which correlate with more established breast cancer prognostic markers. PUBLIC HEALTH RELEVANCE: The study of new tumor suppressors such as Smad 3 may expand the conventional staging and grading of breast cancer by facilitating an organized molecular staging of disease. These efforts will ultimately allow for the further development of individualized prognostic markers to actualize patient-specific treatment strategies. Therapeutic targeting of CDK/cyclin activity to restore Smad 3 tumor suppression may hold promise for patients with cyclin overexpressing and basal-like breast cancers, who currently have the disease sub-type with the worst prognosis.